Perfusion method for manufacturing etanercept

ABSTRACT

Production of etanercept using perfusion methods achieves attractive yields of properly folded protein. Desired temperature, feed media, titers and percent correctly folded protein are disclosed.

FIELD OF THE INVENTION

The present invention relates generally to methods of manufacturingetanercept, and, more particularly, to manufacture of etanercept usingperfusion in which cells producing the protein are cultured in thepresence of a culture medium that is being continuously or periodicallyremoved from and added to a reaction vessel where the production istaking place. The disclosed method achieves production of correctlyfolded etanercept in excellent yields having desired glycosylationprofiles.

BACKGROUND OF THE INVENTION

A form of etanercept commercially available from Amgen under thetrade-name Enbrel® is known to be a dimeric fusion polypeptideconsisting of the extracellular ligand-binding portion of the human 75kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fcportion of human IgG1. It consists of 934 amino acids and has anapparent molecular weight of approximately 150 kilodaltons (PhysiciansDesk Reference, 2002, Medical Economics Company Inc.) The Fc componentof etanercept contains the constant heavy 2 (CH2) domain, the constantheavy 3 (CH3) domain and hinge region, but not the constant heavy 1(CH1) domain of human IgG1. An Fc domain can contain one or all of thedomains described above. Etanercept is usually produced by recombinantDNA technology in a Chinese hamster ovary (CHO) mammalian cellexpression system.

People suffering from certain types of inflammatory diseases such asrheumatoid arthritis, plaque psoriasis, psoriatic arthritis, juvenileidiopathic arthritis, and ankylosing spondylitis, have an immune systemthat over produces tumor necrosis factor (“TNF”). Administration ofetanercept has been found effective for treatment of some inflammatorydiseases because it can reduce the levels of the active form of TNF in asubject by binding to TNF as a decoy receptor.

Etanercept can be produced in a known manner by recombinant DNAtechnology in a Chinese hamster ovary (“CHO”) mammalian cell expressionsystem. Unfortunately, the product that is produced by the CHO cellscontains a large amount of incorrectly or misfolded and/or aggregatedetanercept. For pharmaceutical use, it is desirable to provideetanercept that is relatively free of incorrectly folded and aggregatedprotein because the incorrectly folded/aggregated protein will not havethe same therapeutic effect as the correctly folded protein, and mayactually be detrimental to the patient. Moreover, the known tendency formanufacture of etanercept to cause aggregates or misfolded protein cansignificantly reduce yields and increase costs in recombinant processesused to manufacture the protein. Thus, the alleviation of misfolding isnot only highly desirable from a therapeutic standpoint, but also fromthe standpoint of process economics.

TNFR domain of Etanercept contains several cysteines which formdisulfide bridges. This bridging contributes to protein assuming certainsecondary and tertiary structures (folding). The combination ofpotential bridge forming increases with the number of availablecysteines. Improperly formed cysteine-cysteine bridges result inimproper folding thus lower protein activity. Even when misfolding isthought to be negligible during production of pharmaceutical proteins,e.g., in the case of mammalian secretory expression, aggregation andsome misfolding may still occur.

Need exists for methods capable of producing properly folded etanerceptin commercially attractive yields. Moreover, there exists need forproduction methods which can operate at temperatures other than thosepreviously thought desirable in the art.

SUMMARY OF THE INVENTION

The invention provides a perfusion method for manufacturing etanerceptcomprising the following steps: (a) preparing a mixture comprising cellscapable of expressing a protein comprising etanercept and a culturemedium suitable for conducting such expression; (b) in a suitable vesselcontaining the mixture, causing the cells to produce the proteincomprising etanercept; and (c) periodically or continuously removingspent culture medium from, and adding fresh culture medium to, thereaction vessel. The disclosed method is capable of producing correctlyfolded etanercept having a glycosylation profile substantially similarto that of commercially available etanercept sold under the tradenameEnbrel®. Moreover, the disclosed perfusion method can produce etanerceptprotein in which correctly folded etanercept comprises greater than 40,50 or 60 wt. % of the protein.

The medium in which production is carried out preferably comprisesSFM4CHO® medium or a mixture of BalanCD™ CHO Growth A and HyClone™Hycell CHO mediums (most preferably the BalanCD/Hycell medium mixture)and is supplemented with feeds promoting cell growth and productivity,such as glutamine, CHOZN, Feed 1, Feed 2 and Efficient Feed A (suchfeeds being identified further below). Further supplements present inthe culture medium are selected from the group consisting of galactose,dexamethasone and ManNac which are shown to improve culture efficiencyincluding enhancements from the standpoint of generally achievingdesired glycosylation profile, which involves, in particular, attaininghigher degrees of sialylation in the secreted proteins. In particularlypreferred embodiments of the disclosed method, all three of thesesupplements (galactose, dexamethasone and ManNac) are used.

Cells producing etanercept are present in the vessel at a density of atleast 1,000,000 cells/ml, and preferably at a density of at least5,000,000, and most preferably at least about 10,000,000 cells/ml. Priorto step (a), during a growth phase conducted to increase the number ofcells capable of expressing etanercept (before substantial initiation ofproduction phase), such cells capable of expressing a protein comprisingetanercept can be grown at a temperature selected from; (i) about 28° C.to about 37° C.; and (ii) preferably about 35° C. to about 36° C. Duringa subsequent production phase, after the growth phase is substantiallycompleted, the etanercept production is carried out at a temperatureselected from (i) greater than about 32° C.; (ii) greater than about 33°C.; (iii) greater than about 34° C.; (iv) greater than about 35° C.; (v)the range of about 33° C. to about 36° C.; (vi) the range of about 35°C. to about 36° C.; (vii) 32.5° C.; (viii) 33.5° C.; (ix) 34.5° C.; and(x) 35.5° C. The method of the invention preferably comprisescontinuously or periodically, but preferably continuously, harvestingthe etanercept during the production thereof. Moreover, the removal ofspent medium and replacement with fresh culture medium, i.e., perfusion,occurs preferably continuously. Harvesting of etanercept, present in thecontinuously withdrawn culture medium, is also preferably carried outcontinuously.

A particularly preferred perfusion method for producing correctly foldedetanercept involves conducting the production phase above 33° C. andbetween 33° C. to about 36° C., and most preferably at about 33.5° C.using a culture medium comprising the BalanCD/Hycell mixture (approx.1:1), CHOZN, cottonseed hydrolysate, dexamethasone, galactose andManNAc. Other feed supplements may be present, such as glutamine, Feed1, Feed 2, Efficient Feed A and manganese chloride.

The volumetric productivity of the described process and the quality ofthe produced etanercept can be evaluated by using several methods wellknown to those skilled in the art. These methods include but are notlimited to assays that quantify total and active protein (titers),qualify level of protein sialylation such as the isoelectric focusing(IEF) gels, hydrophobic Interaction chromatography and others.

The invention is further directed to a method for producing etanerceptto reduce protein misfolding comprising culturing a recombinantmammalian host cell which encodes a protein comprising etanercept so asto produce such protein, wherein during a production phase, the hostcell is cultured using a perfusion process at a temperature selectedfrom (i) greater than about 32° C.; (ii) greater than about 33° C.;(iii) greater than about 34° C.; (iv) greater than about 35° C.; (v) therange of about 33° C. to about 36° C.; (vi) the range of about 35° C. toabout 36° C.; (vii) 32.5° C.; (viii) 33.5° C.; (ix) 34.5° C.; and (x)35.5° C.; to obtain a protein product comprising at least about 40 wt.%, preferably at least about 50 wt. %, and most preferably at leastabout 60 wt. % of correctly folded etanercept, preferably as determinedby HIC chromatography, and such that the total amount of protein product(correctly and incorrectly folded protein) is produced in titers of atleast about 0.2 to about 1 g/L. Titers can be measured in a known mannerusing conventional methods such as the ForteBio method.

In a further embodiment the invention provides a perfusion method formanufacturing correctly folded etanercept comprising the followingsteps: (a) preparing a mixture comprising cells capable of expressing aprotein comprising etanercept and a culture medium suitable forconducting such expression; (b) in a suitable vessel containing themixture, causing the cells to produce the protein comprising etanercept;and (c) periodically or continuously removing spent culture medium from,and adding fresh culture medium to, the reaction vessel; and wherein:(1) the culture medium comprises BalanCD/Hycell® medium, dexamethasone,galactose and ManNAc; (2) prior to step (a), the cells capable ofexpressing the protein comprising etanercept are grown in a growth phaseat a temperature of 28° C. to 37° C.; (3) production of the proteincomprising etanercept is carried out at a temperature of 33° C. to 36°C.; and (4) the protein comprising etanercept comprises at least 40 wt.%, 50 wt. %, or 60 wt. % of correctly folded etanercept, and wherein thetotal amount of correctly folded and incorrectly folded protein producedduring the production phase is produced in titers of about 0.2 to about1 g/L.

The present invention is further directed to correctly folded etanerceptproduced by a perfusion method comprising the following steps: (a)preparing a mixture comprising cells capable of expressing a proteincomprising etanercept and a culture medium suitable for conducting suchexpression; (b) in a suitable vessel containing the mixture, causing thecells to produce the protein comprising etanercept; and (c) periodicallyor continuously removing spent culture medium from, and adding freshculture medium to, the reaction vessel; and wherein: (1) the culturemedium comprises the above referenced BalanCD/Hycell medium mixture,dexamethasone, galactose and ManNAc; (2) prior to step (a), the cellscapable of expressing the protein comprising etanercept are grown in agrowth phase at a temperature of 28° C. to 37° C.; (3) production of theprotein comprising etanercept is carried out at a temperature of 33° C.to 36° C.; and (4) the protein comprising etanercept comprises at least40 wt. %, 50 wt. %, or 60 wt. % of correctly folded etanercept, andwherein the total amount of correctly folded and incorrectly foldedprotein produced during the production phase is produced in titers ofabout 0.2 to about 1 g/L.

A further embodiment of the invention is a perfusion method forproducing correctly folded etanercept having a glycosylation profilesubstantially similar to that of commercially available etanercept soldunder the tradename Enbrel®, said method comprising the steps of: (a)preparing a mixture comprising cells capable of expressing a proteincomprising etanercept and a culture medium suitable for conducting suchexpression; (b) in a suitable vessel containing the mixture, causing thecells to produce the protein comprising etanercept; and (c) periodicallyor continuously removing spent culture medium from, and adding freshculture medium to, the reaction vessel; wherein dexamethasone, galactoseand ManNAc are present in the culture medium to achieve saidsubstantially matching glycosylation profile. In a further preferredembodiment, the additional inclusion of cottonseed hydroysates is foundto further enhance the glycoprofile.

Another embodiment of the invention is a perfusion method for producingcorrectly folded etanercept having a glycosylation profile substantiallysimilar to that of commercially available etanercept sold under thetradename Enbrel®, said method comprising the steps of: (a) preparing amixture comprising cells capable of expressing a protein comprisingetanercept and a culture medium suitable for conducting such expression;(b) in a suitable vessel containing the mixture, causing the cells toproduce the protein comprising etanercept; and (c) periodically orcontinuously removing spent culture medium from, and adding freshculture medium to, the reaction vessel; wherein:

(i) dexamethasone, galactose and ManNAc are present in the culturemedium in amounts sufficient to achieve said substantially matchingglycosylation profile and

(ii) the culture medium comprises feed media comprising theabove-mentioned BalanCD/Hycell based medium mixture, CHOZN, glutamineand, optionally, cottonseed hydrolysate in amounts sufficient to achievea production titer of correctly folded and incorrectly folded proteinproduced during the production phase of about 0.2 to about 1 g/L; and

(iii) production of the protein comprising etanercept is carried out ata temperature selected from (i) greater than about 32° C.; (ii) greaterthan about 33° C.; (iii) greater than about 34° C.; (iv) greater thanabout 35° C.; (v) the range of about 33° C. to about 36° C.; (vi) therange of about 35° C. to about 36° C.; (vii) 32.5° C.; (viii) 33.5° C.;(ix) 34.5° C.; and (x) 35.5° C.

In yet a further embodiment, the disclosed method is a perfusion methodfor manufacturing etanercept comprising the following steps: (a)preparing a mixture comprising cells capable of expressing a proteincomprising etanercept and a culture medium suitable for conducting suchexpression; (b) in a suitable vessel containing the mixture, causing thecells to produce the protein comprising etanercept; and (c) periodicallyor continuously removing spent culture medium from, and adding freshculture medium to, the reaction vessel; wherein (i) step (b) is carriedout at or above 33° C.; (ii) the culture medium comprises at least oneof dexamethasone, galactose and ManNAc; (iii) the protein comprisingetanercept comprises at least 60 wt. % correctly folded etanercept; (iv)the protein comprising etanercept is produced in titers of 0.2 to about1 g/L; and (v) the etanercept has a glycosylation profile substantiallysimilar to that of commercially available etanercept sold under thetradename Enbrel®. Preferably all three of the supplements galactose,dexamethasone and ManNAc are present in the culture medium. Aparticularly preferred production temperature is in the range of 33° C.to 34° C.

The present invention can produce correctly folded etanercept inexcellent yields, having desired glycosylation profile necessary fortherapeutic effect, and preferably at production temperatures higherthan those previously thought necessary or desirable in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a process of the present invention.

FIG. 2 shows IEF gels with charge profiles for etanercept samplesproduced using the process of the invention.

FIG. 3 shows N-glycan analysis of etanercept samples produced using theprocess of the invention.

FIG. 4 shows a process of the present invention.

FIG. 5 shows IEF gels with charge profiles for etanercept samplesproduced using the process of the invention.

FIG. 6 shows IEF gels with charge profiles for etanercept samplesproduced using the process of the invention.

FIG. 7 shows N-Glycan analysis of etanercept samples produced using theprocess of the invention. Chromatograms are of innovator protein (panelA), the third medium exchange of Shake Flask 3 (panel B), andappropriate harvest from the fed-batch Control Flask (panel C).

FIG. 8 is a bar graph representing the percent of correctly foldedetanercept produced in the working examples, as determined by HICchromatography (including Enbrel control). Cultures were expanded underthe conditions indicated (growth temperature), and product was produced(production temperature) in shake flasks seeded at the indicatedconcentrations. Bars indicated percent of material in the peakrepresenting correctly folded product from chromatograms such as thoseshown in FIGS. 9 through 13.

FIG. 9 contains HIC chromatograms in which trace A is Enbrel control andtrace B is SF1, medium exchange 3 from FIG. 8. FIGS. 9 through 13 depictthe results of Hydrophobic Interaction Chromatography (HIC) of selectedsamples from media exchange study with respect to etanercept producedaccording to examples 1 and 2. Samples were subjected to HIC analysisafter media exchange or harvest. Clipped, correctly folded, andmisfolded product is indicated for each chromatogram. Integrations ofthe correctly folded peak are represented by the bar chart in FIG. 8.

FIG. 10 contains HIC chromatograms in which trace A is SF2, mediumexchange 3 from FIG. 8; and trace B is SF3, medium exchange 1 from FIG.8.

FIG. 11 contains HIC chromatograms in which trace A is SF3, mediumexchange 3 from FIG. 8; and trace B is SF3, harvest from FIG. 8.

FIG. 12 contains HIC chromatograms in which trace A is SF4, mediaexchange 3 from FIG. 8; and trace B is SF5, media exchange 3 from FIG.8.

FIG. 13 contains HIC chromatograms in which trace A is SF6, mediaexchange 3 from FIG. 8; and trace B is control shake flask, sampled atthe media exchange 3 time point as represented in FIG. 8.

FIG. 14 contains the viable cell density, perfusion rate, and specificperfusion rate from the perfusion bioreactor of Example 3.

FIG. 15 contains the titer and specific productivity of the perfusionculture of Example 3.

FIG. 16 contains HIC chromatograms in which trace A is Enbrel control,trace B is a sample from the harvest of a fed-batch bioreactor, trace Cis from day 9 of the perfusion bioreactor in FIG. 14, and trace D isfrom day 12 of the perfusion bioreactor in FIG. 14.

FIG. 17 contains the N-glycan chromatograms of Enbrel reference (A), asample of CHS-0214 from day 9 of the perfusion bioreactor (B), and asample of CHS-0214 from day 12 of the perfusion sample (C) shown in FIG.14.

FIG. 18 is a chart of culture viable cell density (VCD) for mediaformulations of Example 4.

FIG. 19 is a chart of viability for media formulations of Example 4.

FIG. 20 is a photograph of isoelectrofocusing (IEF) Gel for mediaformulations of Example 4.

FIG. 21 is a titer graph for media formulations of Example 4.

FIG. 22A is a chart of culture viable cell density (VCD) for mediaformulations of Example 5.

FIG. 22B is a chart of viability for media formulations of Example 5.

FIG. 23A is a chart of culture viable cell density (VCD) for mediaformulations of Example 6.

FIG. 23B is a chart of viability for media formulations of Example 6.

FIG. 24 is a photograph of isoelectrofocusing (IEF) Gel for mediaformulations of Example 6.

FIG. 25 is a photograph of isoelectrofocusing (IEF) Gel for mediaformulations of Example 7.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. Synonyms for certain termsare provided. A recital of one or more synonyms does not exclude the useof other synonyms. The use of examples anywhere in this specificationincluding examples of any terms discussed herein is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term. The invention is not limited to the variousembodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

The term “etanercept” as used herein refers to a polypeptide which is adimeric fusion polypeptide consisting of the extracellularligand-binding portion of the human 75 kilodalton (p75) tumor necrosisfactor receptor (TNFR) linked to the Fc portion of human IgG1. Itconsists of 934 amino acids and has an apparent molecular weight ofapproximately 150 kilodaltons. For the purposes of the presentapplication, the term “etanercept” also encompasses etanercept withminor modifications in the amino acid structure (including deletions,additions, and/or substitutions of amino acids) which do notsignificantly affect the function, potency, or avidity of etanercept.The term “etanercept” encompasses all forms and formulations of Enbrel®,including but not limited to concentrated formulations, injectableready-to-use formulations; formulations reconstituted with water,alcohol, and/or other ingredients, and others. The term etanercept isalso intended to include biosimilar or biobetter variants of theetanercept used in commercial Enbrel®. For example, a biosimilar orbiobetter of etanercept may have a slightly different glycosylationprofile than commercial Enbrel®. In addition a biosimilar or biobettervariant of the etanercept preparation found in commercial Enbrel® mayexhibit a reduction in the amount of aggregates/misfolds present alongwith the active, properly folded etanercept ingredient.

The term “correctly folded etanercept” as used herein is intended todenote a folding conformation of the etanercept homodimer (as definedabove) having biological activity for inhibition of TNF and conformationthat are the same or substantially the same as the conformation andbiological activity of the active ingredient in Enbrel®.

The term “incorrectly folded etanercept” as used herein is intended toencompass: (i) a homodimeric protein having the same amino acid sequenceas etanercept (as defined above), but having a conformation differentfrom that of correctly folded etanercept, wherein said differentconformation renders the protein lacking or substantially lacking inbiological activity as a TNF inhibitor; and/or (ii) an aggregate inwhich two or more correctly and/or incorrectly folded etanercepthomodimers have become associated (i.e., aggregated or clumped) in sucha manner as to form species having higher molecular weight thancorrectly folded etanercept; and/or (iii) a mixture of (i) and (ii);and/or (iv) aggregated i.e., clumped protein compositions comprising thesame or essentially the same sequence, or portions thereof, as correctlyfolded etanercept but which exhibit decreased elution position (due togreater hydrophobicity) on an HIC column as compared to correctly foldedetanercept.

The term “growth phase” denotes a phase in which cells capable ofexpressing etanercept are generally first cultured at a temperaturewhich promotes exponential logarithmic growth of the cells prior toentering into the production phase. A suitable temperature for thegrowth phase is generally in the range of 34° C. to about 38° C. asdescribed in U.S. Pat. No. 7,294,481.

The term “production phase is understood to have the same meaning asthat ascribed in U.S. Pat. No. 7,294,481, incorporated by referenceherein in its entirety. In particular, the term refers to the periodduring which cell growth has plateaued, i.e., logarithmic cell grown hasended, and protein production is primary. According to the presentinvention, the production phase is carried out under perfusionconditions, preferably at a temperature in the range of about 32.5° C.to about 37° C., and preferably in the range of about 33.5° C. to about35.5° C.

Perfusion has the meaning generally explained below and can also bebriefly understood as a method of culture in which waste medium (spentmedium) is removed from the culture and the displaced medium isreplenished with fresh medium. This may preferably be done in acontinuous manner, but may also be performed in a stepwise discontinuousmanner in which spent medium is replaced with fresh medium at desiredintervals prior to completion of the production phase. The addition offresh medium and elimination of waste products provides the cells withan environment that is better suited to achieving and maintaining highcell concentrations with higher productivity.

The term “treatment” refers to any administration or application ofremedies for disease in a mammal and includes inhibiting the disease,arresting its development, relieving the disease (for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function) or stimulating an inefficient process. The termincludes obtaining a desired pharmacologic and/or physiologic effect andcovering any treatment of a pathological condition or disorder in amammal. The effect may be prophylactic in terms of completely orpartially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse effect attributable to the disorder. It includes (1) preventingthe disorder from occurring or recurring in a subject who may bepredisposed to the disorder but is not yet symptomatic, (2) inhibitingthe disorder, such as arresting its development, (3) stopping orterminating the disorder or at least its associated symptoms, so thatthe host no longer suffers from the disorder or its symptoms, such ascausing regression of the disorder or its symptoms, for example, byrestoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain and/or tumorsize.

The term “pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material,formulation auxiliary, or excipient of any conventional type. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation.

The term “composition” or “formulation” refers to a mixture that usuallycontains a carrier, such as a pharmaceutically acceptable carrier orexcipient that is conventional in the art and which is suitable foradministration into a subject for therapeutic, diagnostic, orprophylactic purposes. It may include a cell culture in which thepolypeptide or polynucleotide is present in the cells or in the culturemedium. For example, compositions for oral administration can formsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations, oral rinses or powders.

The terms “BalanCD/Hycell” denotes a mixture (approx. 1:1) of thecommercially obtainable feeds sold as BalanCD™ CHO Growth A and HyClone™Hycell CHO as referenced in the Table, below.

The following Table is a listing of the commercially available feeds andfeed supplements useful in the present invention.

Vendor Stock Raw Material Catalog Conc Description Source NumberCategory (g/L) Useful Range Use Notes BalanCD ™ CHO Irvine 94120-10LBase medium 23.725 n.a. base Growth A Scientific medium HyClone ™ HyCellThermo SH30933 Base medium 25.400 n.a. base CHO Scientific mediumHyClone ™ Thermo SH30518.04 Base medium 19.830 n.a. used in SFM4CHOScientific seed train; D-(+)-Galactose SAFC G5388 Glycan feed ≦10 mMused at 10 mM final; to optimize product quality Dexamethasone SAFCD4902 Glycan feed  ≦1 uM used at 0.8-1.0 uM; to optimize product qualityManNAc SAFC A8176 Glycan feed ≦20 mM used at 10-20 mM (N- final;acetylmannosamine) to optimize product quality BalanCD ™ CHO Irvine94119-10L Titer feed 55.776 10% (v/v) Boosts titer Feed 1 Scientificwhen added alone or with CHOZN BalanCD(tm) CHO Irvine 94121 Titer feedFeed 2 Scientific HyClone ™ Cell Thermo SH30865.04 Titer feed 50 10-20%(v/v) Used in Boost 5 Scientific control experiments CHO CD LifeA1023401 Titer feeds Boosts titer EfficientFeed A Technologies whenadded alone or with with CHOZN Cottonseed FrieslandCampina CNE50M-UFTiter feed 100 15% (v/v) increases Hydrolysate Domo cell growth (“CSH”)and specific productivity EX-Cell CHOZN SAFC 24331C-10L Titer feed 5010-20% (v/v) complex Platform Feed feed; boosts titer when added aloneor in combination with other complex feedsPerfusion-Based Manufacture of Etanercept

The present invention provides methods of manufacturing etanercept whichinvolve the use of perfusion. The term “perfusion” as used herein isintended to generally denote a process in which a suspension cellculture is continuously or periodically, and most preferablycontinuously, supplied with fresh medium to a bioreactor while spentculture media is continuously removed, i.e., harvested (preferably withthe product) in order that product contained therein can be continuouslyharvested, and the waste and toxic materials present in the spent mediumcan be removed from the bioreactor. Using appropriate filtration meanswell known in the art, the cells are then continuously filtered from theharvest stream and returned to the bioreactor to maintain a constantculture volume. Such a process, typically carried out continuously,allows the cells to reach high densities. Accordingly, densities as highas 15-20 million cells/mL can routinely be reached and maintained forextended periods of time, e.g. at least two weeks. This can result invery highly productive cell culture process that can produce for alonger period of time as opposed to batch or fed-batch cultures.Alternatively, rather than continuously harvesting product from theremoved spent medium, the product can be maintained and concentrated inthe culture, and then harvested periodically, or at the end of theculture. Utilization of appropriate size filters can allow for removalof only waste, with retention of the recombinant product in thebioreactor culture. In such a process, sometimes referred to as “extremedensity” or XD process, the product can be harvested periodically or atthe end of the culture.

We have now found that a predetermined glycoprofile of etanerceptproduced in a perfusion process can be achieved when the culture mediumcomprises at least one of dexamethasone, galactose and ManNAc, and mostpreferably when all three are present in the culture medium. Suitableamounts are referenced in the Examples below. We have also discoveredthat the additional presence of cottonseed hydroysates in such perfusionprocess can further enhance the glycoprofile. The term “glycoprofile” or“glycosylation profile” are well understood in the art, and should beunderstood to include the level or degree of sialylation occurring onthe glycan groups attached to the etanercept protein.

Example 1

A shake flask format is used to investigate processing conditionssimilar and comparable to a perfusion process. High density shake flaskcultures (5 million cells per milliliter to 20 million cells permilliliter) are established from cultures expanded at temperatures inthe range of about 35° C. to 37° C. in SFM4CHO medium supplemented withCell Boost 5 feed and about 0.5 uM-1 uM dexamethasone.

Example 1 Media Formulation

Feed Component Concentration SFM4CHO 1x Cell Boost 5 20% Dexamethasone0.5 uM

Each culture, maintained in temperatures ranging from 32° C. to 35.5°C., was allowed to produce Etanercept protein for two days before mediumwas fully exchanged for a subsequent round of production. These 2-dayharvest intervals are comparable to a perfusion rate of 0.5 bioreactorvolume per day. The medium exchange is repeated 4 times (4 cycles).Harvested media is frozen at −80° C. Titers are analyzed by ForteBio andTNF-binding ELISA. Additionally each sample is assessed for N-linkedglycoprofile, protein charge distribution by IEF gel and for proteinfolding by hydrophobic interaction chromatography (HIC).

In order to support the high cell numbers necessary for inoculation ofhigh density production cultures typically achieved in a perfusionprocess, the seed train is conducted in large volume shake flasksmaintained at 35° C. or 37° C., 5% CO2 level and the speed of theorbital shaker is adjusted to 125 rpm. Production phase shake flaskscontaining SFM4CHO medium supplemented with Cell Boost 5 feed and 0.5 uMdexamethasone are inoculated at cell densities either 10 million cellsper milliliter or 20 million cells per milliliter. The production phaseis conducted at a temperature in the range of about 32° C. to about 36°C., otherwise all other culture conditions are the same. Cultures aremonitored daily for viable cells densities and viabilities. Toinvestigate reactor volume exchange conditions comparable to a perfusionrate of 0.5 bioreactor volume per day, the medium in each culture wasfully exchanged every 48 hours. The harvested and clarified media arefrozen at −80° C. Following each spent medium harvest, cells areresuspended in fresh medium and allowed to accumulate recombinantproduct for another 48 hours, the aforementioned process being repeatedfor a total of 4 cycles. At the conclusion of the experiment all samplesare thawed and analyzed with respect to charge profile (byisoelectrofocusing gels, IEF), N-glycan profile and titers. A controlexperiment was conducted using a fed-batch culture inoculated at 0.4million cells per milliliter in SFM4CHO medium supplemented with CellBoost 5 feed and 0.5 uM dexamethasone. The conditions for the controlexperiment involved an expansion phase at 35° C. and the productionphase at 32° C. initiated on day 5. The Etanercept protein produced inthe control experiment is allowed to accumulate without medium exchangefor the length of the experiment. Samples from the control culture arewithdrawn every 48 hours during the production phase, frozen at −80° C.,and analyzed along with the remaining experimental samples. Theexperimental design of the experiments conducted according to thisExample 1 is depicted in FIG. 1. The charge profile of etanerceptproduced in this Example is shown in FIG. 2. FIG. 3 shows the N-Glycananalysis of Gel #7 and Gel #10 of FIG. 2. Etanercept protein produced inthis example using a medium exchange technique designed to simulateperfusion processing elicits a similar profile to that of the innovatorbased on charge profile assessed by IEF gel and titers (FIG. 2). TheN-glycan distribution shown by chromatograms in FIG. 3 has also similarprofile to the reference standard. Based on the productivitiesdetermined to be approximately 0.3 g/L from culture at cell density of10 million cells per milliliter, we expect the disclosed method toachieve production of approximately 0.75 to 1 g/L per day based onculture at expected minimum density of 50 million cells per milliliter.

Example 2

In order to support the high cell numbers necessary for inoculation ofhigh density production cultures characteristic of perfusion processes,the seed train is conducted in large volume shake flasks maintained at35° C. or 37° C., 5% CO2 level and the speed of the orbital shaker isadjusted to 125 rpm. Production phase shake flasks containing SFM4CHOmedium supplemented with Cell Boost 5 feed and 0.5 uM dexamethasone areinoculated at cell densities either 5 million cells per milliliter or 8million cells per milliliter. The following media formulation was used.

Example 2 Media Formulation

Feed Component Concentration SFM4CHO 1x Cell Boost 5 20% Dexamethasone0.5 uM

The production phase is conducted at temperatures 33.5° C. or 35.5° C.,otherwise all other culture conditions are the same. Cultures aremonitored daily for viable cells densities and viabilities. To achievethe equivalent of a perfusion rate of 0.5 bioreactor volume per day themedium in each culture is fully exchanged every 48 hours. The harvested,clarified spent media is frozen at −80° C. Following each spent mediumharvest, cells were resuspended in fresh medium and allowed toaccumulate recombinant product for another 48 hours; with theaforementioned process being repeated for a total of 5 cycles. At theconclusion of the experiment, all samples were thawed and analyzed withrespect to titers, charge profile (by isoelectrofocusing gels, IEF),N-glycan profile and folding. Control conditions involved fed-batchculture inoculated at 0.4 million cells per milliliter in SFM4CHO mediumsupplemented with Cell Boost 5 feed and 0.5 uM dexamethasone. Thecontrol conditions involved the expansion phase conducted at 35° C. andthe production phase at 35.5° C. initiated on day 5. The Etanerceptprotein was allowed to accumulate without medium exchange for the lengthof the experiment. Samples from the control culture were withdrawn every48 hours during the production phase, frozen at −80° C., and analyzedalong with the remaining experimental samples. The experimental designof the experiments conducted according to this Example 2 is depicted inFIG. 4. FIG. 5 shows the IEF gels for etanercept harvested after mediumexchange #1 (i.e., 2 days after initiation of production phase). IEFgels in FIG. 5 show charge profile of Etanercept proteins produced afterfirst medium exchange (equivalent of day 2 of a continuous perfusion)similar to that of Enbrel®. The control sample which is at this pointstill at low cell density shows similar profile. FIG. 6 shows the IEFgels for etanercept harvested after medium exchange #3 (6 days afterinitiation of production phase). The charge profile of Etanerceptproteins produced after third medium exchange (equivalent to day 6 of acontinuous perfusion) is similar to that of Enbrel®. The desiredisoforms are enclosed by a red box. Control sample shows somedeterioration of product quality as shown by higher content ofundersialylated, higher pI protein species. The data in this Examplelends support to a conclusion that a perfusion system can provide abetter protein quality than the fed-batch culture. Samples from eachmedium exchange are subjected to N-glycan analysis (Melmer et al., AnalBioanal Chem (2010) 398:905-914, HILIC analysis of fluorescence-labeledN-glycans from recombinant biopharmaceuticals). Briefly, glycans arereleased from the test material, labeled with a fluorescent moiety topermit detection, and fractionated by normal-phase HPLC. Chromatogramsof innovator protein (panel A), the third medium exchange of Shake Flask3 (panel B), and appropriate harvest from the fed-batch Control Flask(panel C) are shown in FIG. 7.

Example 3

The seed train is expanded in large-volume shake flasks at 35° C. inSFM4CHO. The production bioreactor is inoculated at seeding densities offrom 1 to 5×10⁶ cells/mL in SFM4CHO containing Cell Boost 5 0.5 uMdexamethasone Table 1), and maintained at temperatures from 33.5° C. to35° C. The media formulation was as follows:

Example 3 Media Formulation

Feed Component Concentration SFM4CHO 1x Cell Boost 5 20% Dexamethasone0.5 uM

An ATF™ cell retention device (Refine Technology) is used to recirculatemedium (containing waste products and desired product) past a hollowfiber filter, with recirculation rates from 0.1 to 2.0 working culturevolumes per minute. The culture is expanded for 0 to 2 days, and thenperfusion is initiated at rates from 0.2 to 2 culture volumes per day.New medium is added as spent medium, containing the product, isharvested through a 0.2 urn pore size hollow fiber filter. Harvestedfluid is chilled to 2-8° C., purified by capture on protein A resin.Aliquots are analyzed for titer and N-glycan distribution, as describedfor Examples 1 and 2. HIC analysis may be used to evaluate the relativeamounts of properly folded etanercept, versus improperlyfolded/aggregated (inactive) material.

FIG. 14 shows the VCD, which reached around 12×10⁶ cells/mL during theperfusion production phase. The perfusion rate, which began at 0.5volumes of medium added per bioreactor volume per day (WD) and increasedto 1.0 VVD when the VCD reached its plateau. The specific perfusion rate(mL of media per million cells per day) ranged from 0.06 to 0.08 duringthe production phase. The titer in samples taken daily was 250 to 350mg/L, while the specific productivity was 15 to 30 pg per cell per day(FIG. 15).

Analysis of correct folding, using HIC, shows that etanercept-containingmaterial from the perfusion bioreactor has a higher percentage ofcorrectly folded etanercept than that produced in a fed-batch culture(compare FIG. 16 panel B, fed-batch, to panels C and D, perfusion).

N-glycan analysis shows the close agreement between etanercept producedin a perfusion bioreactor and Enbrel® reference, as shown in thechromatograms in FIG. 17.

Example 4

Cells were inoculated at 25 million cells per milliliter of media intotwo different base media, SFM4CHO or BalanCD/Hycell, each supplementedwith Cell Boost (in the case of SFM4CHO) or CHOZN feeds (in the case ofBalanCD/Hycell) at final concentration of 10% or 20%. The feeds alsoincluded other supplements that can promote sialylation, i.e.,dexamethasone, galactose and ManNAc. Cottonseed hydrolysates andgalactose were also added to the BalanCD/Hycell-containing medium (seeformulation summaries below)

Example 4 (SF1)

Feed Component Concentration SFM4CHO 1x Cell Boost 5 10% Dexamethasone0.8 uM

Example 4 (SF2)

Feed Component Concentration SFM4CHO 1x Cell Boost 5 20% Dexamethasone0.8 uM

Example 4 (SF3)

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN  10% Cotton SeedHydrolysate 7.5% Galactose 10 mM ManNAc 10 mM

Example 4 (SF4)

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN  20% Cotton SeedHydrolysate 7.5% Galactose 10 mM ManNAc 10 mM

Cultures were maintained at a temperature of 33.5° C. while perfusionwas carried out by exchanging media every 48 hours. Samples from eachmedium exchange were analyzed with respect to titers, isoform profile byIEF gels and for amino acid depletion profile (spent medium analysis).Culture viable cell density (VCD) and viability are shown in FIGS. 18and 19, respectively, while isoform profile correlating with the levelof a sialic acid content is shown in FIG. 20 (IEF Gel for Example 4).The titers were evaluated by ForteBio analysis and are displayed in FIG.21 (Titer Graph for Example 4).

FIGS. 18 and 19 show viable cell density (VCD) and viability profile ofcultures maintained in the four above referenced media formulations (SF1to SF4). BalanCD/Hycell medium supplemented with CHOZN (10% or 20%) andcottonseed hydrolysate, galactose and ManNAc supports better nutritionresulting in superior viability, VCD, product quality (see FIG. 20) andtiters (see FIG. 21).

FIG. 20 shows isoform profile of proteins isolated from culturescultivated in SFM4CHO medium (lanes 2, 3, 7, 8) or in BalanCD/Hycellmedium (lanes 4, 5, 9, 10). Proteins isolated from SFM4CHO cultures showreduced sialylation compared to reference standard (lane 6) whileproteins isolated from BalanCD/Hycell cultures display isoform profilebased on sialic acid content closely matching that of the referencestandard.

FIG. 21 shows that cultures from SFM4CHO medium (P1-1, P1-2, P2-1, P2-2)displayed lower productivity than those cultivated in BalanCD/Hycell 1:1mixture of media (P1-3, P1-4, P2-3, P2-4).

Example 5

Given our desire to develop feeds that will support higher densityperfusion processes, this example contains experimental results fromevaluating various feeds and feed combinations to identify those whichwould provide nutritional support for cultures exceeding 30 millioncells per milliliter, preferably supporting perfusion runs at 50 millioncells per milliliters of culture. Cultures were inoculated at 40 millioncells per milliliter into the BalanCD/Hycell base medium reported inExample 4 above, supplemented with CHOZN (10%) and Feed1 (10%). One ofthe cultures was additionally supplemented with 7.5% cottonseedhydrolyzate. Media composition is provided below.

Example 5 Medium 1

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10% FEED 1 10%L-Glutamine  8 mM Galactose 10 mM

Example 5 Medium 2

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10% FEED 1 10%L-Glutamine  8 mM Galactose 10 mM Cotton seed hydrolysate 7.5% 

The Example 5 Formulations were tested in batch mode using culturelongevity as the end point. Cultures were maintained at 33.5° C. withoutadditional feeding until the viability declined to ˜80%. Viable celldensity and viability of culture are shown in FIGS. 22A and 22B,respectively.

FIGS. 22A and 22B depict viable cell density (VCD) and viability profileof cultures maintained in two different media formulations of theBalanCD/Hycell 1:1 mixture base medium. Both feed formulations resultedin batch culture longevity of 4 days (Any reduction of cell density wasdue to intensive sampling from small volume cultures and attachment ofcells cultivated in high density culture to the pipette)

This Example 5 demonstrates that the BalanCD/Hycell media formulationsdescribed here are rich enough to support high density perfusion runs,and further, that the perfusion rate may perhaps be reduced due todecreased risk of nutrient depletion.

Example 6

Exploring further performance of media formulations described in Example5 on product quality in simulated perfusion mode cultures were set up at40-50 million cells per milliliter in BalanCD/Hycell base mediumsupplemented with 10% CHOZN, 10% Feed1, 10 mM Galactose and 7.5% cottonseed hydrolysate. Additionally two of the three cultures weresupplemented with 0.8 uM dexamethasone and 20 mM ManNac. One of thesetwo cultures received 0.01 uM magnesium chloride. Media composition isprovided in Example 6 Medium 1-3 (see tables below).

Example 6 Medium 1

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10% FEED 1 10%L-Glutamine  8 mM Galactose 10 mM Cotton seed hydrolysate 7.5% 

Example 6 Medium 2

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10% FEED 1 10%L-Glutamine  8 mM Galactose 10 mM Cotton seed hydrolysate 7.5% Dexamethasone 0.8 uM   ManNac 20 mM

Example 6 Medium 3

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10% FEED 1 10%L-Glutamine  8 mM Galactose 10 mM Cotton seed hydrolysate 7.5% Dexamethasone 0.8 uM   ManNAc 20 mM Magnesium chloride 0.01 uM  

Cultures were cultivated at 33.5° C. Perfusion was carried out byperforming medium exchange every 24 hours.

Samples were analyzed with respect to growth (viable cell density andviability), titers and isoform profile using IEF gels. Culture viablecell density (VCD) and viability are shown in FIGS. 23A and 23B,respectively while isoform profile correlating with the level of sialicacid content is shown in FIG. 24 (the IEF Gel for this Example).

FIGS. 23A and 23B show viable cell density (VCD) and viability profileof cultures maintained in three different media formulations of theBalanCD/Hycell base medium. All feed formulations were tested for 5 dayswith four medium exchanges performed every 24 hours. Decrease in celldensity at later stage of the cultures was the result of heavy sampletesting and cells attaching to the pipette.

FIG. 24 shows the isoform profile of samples from harvest culture (day4, fourth medium exchange) composed of Medium 1 (lane 6), Medium 2 (lane7) and Medium 3 (lane 8). Lanes 5 and 9 correspond to referencestandard.

Data generated from experiments described in Example 6 indicates thatdespite similar culture performance with respect to viability and viablecell density, the product quality is further improved by formulation ofMedium 2 and Medium 3.

Example 7

In yet a further example of high density cultures tested in a perfusionprocess involving repetitive medium exchanges at predetermined timeintervals, we inoculated cells at 40-50 million cells per milliliterinto four different BalanCD/Hycell 1:1 mixtures. Mixture 1 wassupplemented with Ex-Cell CHOZN Platform Feed and BalanCD Feed1, Mixture2 was supplemented with Ex-Cell CHOZN Platform Feed, BalanCD Feed1 andBalanCD Feed2; Mixture 3 was supplemented with Ex-Cell CHOZN PlatformFeed and Efficient Feed A; and Mixture 4 was supplemented with Ex-CellCHOZN Platform Feed, BalanCD Feed1, BalanCD Feed2 and Efficient Feed A.All four of the BalanCD/Hycell 1:1 mixtures contained additionalsupplementation of 8 mM L-glutamine, 10 mM galactose, 7.5% cotton seedhydrolysate, 0.8 uM dexamethasone, and 20 mM ManNAc.

Example 7—Medium 1

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10% FEED 1 10%L-Glutamine  8 mM Galactose 10 mM Cotton seed hydrolysate 7.5% Dexamethasone 0.8 uM   ManNAc 20 mM

Example 7—Medium 2

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10%  FEED 1 5%FEED 2 5% L-Glutamine  8 mM Galactose 10 mM Cotton seed hydrolysate7.5%   Dexamethasone 0.8 uM   ManNAc 20 mM

Example 7—Medium 3

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10% FEED 2 10%L-Glutamine  8 mM Galactose 10 mM Cotton seed hydrolysate 7.5% Dexamethasone 0.8 uM   ManNAc 20 mM

Example 7—Medium 4

Feed Component Concentration BalanCD/Hycell 1:1 CHOZN 10%  FEED 1 3%FEED 2 3% Efficient FEED A 3% L-Glutamine  8 mM Galactose 10 mM Cottonseed hydrolysate 7.5%   Dexamethasone 0.8 uM   ManNAc 20 mM

Cultures were maintained at 33.5° C. Perfusion conditions were achievedby replacing medium every 24 hours (for a total of five exchanges).Product quality was measured by IEF gel analysis during four consecutivemedium exchanges (See FIG. 25).

FIG. 25 shows samples form the last of the five medium exchanges (day5): standard is lane 4; samples from Medium 1, Medium 2, Medium 3,Medium 4 (lanes 5-8, respectively). This data indicate that all fourmedia formulations support high cell density perfusion with simulatedperfusion rate of 1 bioreactor volume per day, and that these conditionsallow for production of etanercept protein with sialic acid contentresulting in the isoform distribution substantially similar to thereference standard (commercially available Enbrel®). In a perfusion modewhere the steady medium flow delivers nutrients and removes wastes in acontinuous fashion the product quality can be expected to improve evenfurther.

What is claimed is:
 1. A perfusion method for manufacturing correctlyfolded etanercept comprising the following steps: (a) preparing amixture comprising cells capable of expressing a protein comprisingetanercept and a culture medium suitable for conducting such expression;(b) in a suitable reaction vessel containing the mixture, causing thecells to produce the protein comprising etanercept; and (c) periodicallyor continuously removing spent culture medium from, and adding freshculture medium to, the reaction vessel, wherein the protein comprisingetanercept produced in the method comprises at least 40 wt. %, 50 wt. %,or 60 wt. % of correctly folded etanercept, and wherein the culturemedium comprises a Chinese hamster ovary cell medium as a base feedmedium and wherein the culture medium comprises dexamethasone, galactoseand N-acetylmannosamine (ManNAc).
 2. A perfusion method formanufacturing correctly folded etanercept comprising the followingsteps: (a) preparing a mixture comprising cells capable of expressing aprotein comprising etanercept and a culture medium suitable forconducting such expression; (b) in a suitable reaction vessel containingthe mixture, causing the cells to produce the protein comprisingetanercept; and (c) periodically or continuously removing spent culturemedium from, and adding fresh culture medium to, the reaction vessel;and wherein: (1) the culture medium comprises a Chinese hamster ovarycell medium, dexamethasone, galactose and N-acetylmannosamine (ManNAc);(2) prior to step (a), the cells capable of expressing the proteincomprising etanercept are grown in a growth phase at a temperature of28° C. to 37° C.; (3) production of the protein comprising etanercept iscarried out at a temperature of 33° C. to 36° C.; and (4) the proteincomprising etanercept comprises at least 40 wt. %, 50 wt. %, or 60 wt. %of correctly folded etanercept, and wherein the total amount ofcorrectly folded and incorrectly folded protein is produced at a titerof about 0.2 to about 1 g/L.
 3. The perfusion method of claim 2 whereinthe production of the protein comprising etanercept is conducted at 33°C. to 34° C., and the amount of correctly folded etanercept is at least60 wt. %.
 4. The perfusion method of claim 2 in which an alternatingtangential flow cell retention device is used to recirculate mediumcontaining waste products and the protein comprising etanercept past ahollow fiber filter whereby the waste products and the proteincomprising etanercept are removed from the reaction vessel.
 5. Theperfusion method of claim 2 wherein the culture medium further comprisesat least one feed supplement selected from glutamine and cottonseedhydrolysate.
 6. The perfusion method of claim 5 wherein the culturemedium comprises cottonseed hydrolysate.
 7. A perfusion method forproducing correctly folded etanercept said method comprising the stepsof: (a) preparing a mixture comprising cells capable of expressing aprotein comprising etanercept and a culture medium suitable forconducting such expression; (b) in a suitable reaction vessel containingthe mixture, causing the cells to produce the protein comprisingetanercept; and (c) periodically or continuously removing spent culturemedium from, and adding fresh culture medium to, the reaction vessel;wherein dexamethasone, galactose and N-acetylmannosamine (ManNAc) arepresent in the culture medium.
 8. The perfusion method of claim 7wherein (1) prior to step (a), the cells capable of expressing theprotein comprising etanercept are grown in a growth phase at atemperature selected from (i) about 28° C. to about 37° C.; and (ii)about 35° C. to about 36° C.; and (2) production of the proteincomprising etanercept is carried out at a temperature selected from (i)greater than about 32° C.; (ii) greater than about 33° C.; (iii) greaterthan about 34° C.; (iv) greater than about 35° C.; (v) the range ofabout 33° C. to about 36° C.; (vi) the range of about 35° C. to about36° C.; (vii) 32.5° C.; (viii) 33.5° C.; (ix) 34.5° C.; and (x) 35.5° C.9. The method of claim 8 wherein the protein comprising etanerceptproduced in the method comprises at least 40 wt. %, 50 wt. %, or 60 wt.% of correctly folded etanercept; the culture medium comprises a Chinesehamster ovary cell base medium, glutamine and cottonseed hydrolysate;and correctly folded and incorrectly folded protein is produced at atiter of about 0.2 to about 1 g/L.
 10. A perfusion method for producingcorrectly folded etanercept, said method comprising the steps of: (a)preparing a mixture comprising cells capable of expressing a proteincomprising etanercept and a culture medium suitable for conducting suchexpression; (b) in a suitable reaction vessel containing the mixture,causing the cells to produce the protein comprising etanercept; and (c)periodically or continuously removing spent culture medium from, andadding fresh culture medium to, the reaction vessel; wherein: (i)dexamethasone, galactose and N-acetylmannosamine (ManNAc) are present inthe culture medium and (ii) the culture medium comprises feed mediacomprising a Chinese hamster ovary cell medium, glutamine and cottonseedhydrolysate, and correctly folded and incorrectly folded protein isproduced at titer of about 0.2 to about 1 g/L; and (iii) production ofthe protein comprising etanercept is carried out at a temperatureselected from (i) greater than about 32° C.; (ii) greater than about 33°C.; (iii) greater than about 34° C.; (iv) greater than about 35° C.; (v)the range of about 33° C. to about 36° C.; (vi) the range of about 35°C. to about 36° C.; (vii) 32.5° C.; (viii) 33.5° C.; (ix) 34.5° C.; and(x) 35.5° C.
 11. The method of claim 10 wherein the production of theprotein comprising etanercept is carried out at a temperature of 33° C.to 36° C., and the protein comprising etanercept comprises at least 60wt. % correctly folded etanercept.
 12. The method of claim 11 whereinthe production of the protein comprising etanercept is carried out at atemperature of 33° C. to 34° C.
 13. A perfusion method for manufacturingcorrectly folded etanercept comprising the following steps: (a)preparing a mixture comprising cells capable of expressing a proteincomprising etanercept and a culture medium suitable for conducting suchexpression; (b) in a suitable reaction vessel containing the mixture,causing the cells to produce the protein comprising etanercept; and (c)periodically or continuously removing spent culture medium from, andadding fresh culture medium to, the reaction vessel; wherein (i) step(b) is carried out at or above 33° C.; (ii) the culture medium comprisesat least one of dexamethasone, galactose, glutamine, cottonseedhydrolysate, and N-acetylmannosamine (ManNAc); (iii) the proteincomprising etanercept comprises at least 60 wt. % correctly foldedetanercept; and (iv) the protein comprising etanercept is produced at atiter of about 0.2 to about 1 g/L.
 14. The perfusion method of claim 13wherein the culture medium comprises dexamethasone, galactose andManNAc.
 15. The perfusion method of claim 14 wherein the culture mediumcomprises a Chinese hamster ovary cell medium.
 16. A perfusion methodfor manufacturing correctly folded etanercept comprising the followingsteps: (a) preparing a mixture comprising cells capable of expressing aprotein comprising etanercept and a culture medium suitable forconducting such expression; (b) in a suitable reaction vessel containingthe mixture, causing the cells to produce the protein comprisingetanercept; and (c) periodically or continuously removing spent culturemedium from, and adding fresh culture medium to, the reaction vessel,wherein the protein comprising etanercept produced in the methodcomprises at least 40 wt. %, 50 wt. %, or 60 wt. % of correctly foldedetanercept, wherein the culture medium comprises a Chinese hamster ovarycell medium as a base feed medium and wherein the culture mediumcomprises cottonseed hydrolysate.